HCV E1E2 vaccine compositions

ABSTRACT

HCV E1E2 compositions comprising E1E2 antigens, submicron oil-in-water emulsions and/or immunostimulatory nucleic acid sequences are described. The compositions can be used in methods of stimulating an immune response in a vertebrate subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/187,257, filed Jun. 28, 2002, which is related to provisional patentapplication No. 60/302,227, filed Jun. 29, 2001, from which applicationpriority is claimed under 35 USC '119(e)(1), and which applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention pertains generally to vaccine compositions. Inparticular, the invention relates to HCV E1E2 vaccine compositionscomprising E1E2 antigens, submicron oil-in-water emulsions and/or CpGoligonucleotides.

BACKGROUND OF THE INVENTION

Hepatitis C Virus (HCV) is the principal cause of parenteral non-A,non-B hepatitis (NANBH). The virus is present in 0.4 to 2.0% of blooddonors. Chronic hepatitis develops in about 50% of infections and ofthese, approximately 20% of infected individuals develop liver cirrhosiswhich sometimes leads to hepatocellular carcinoma. Accordingly, thestudy and control of the disease is of medical importance.

HCV was first identified and characterized as a cause of NANBH byHoughton et al. The viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kbpositive-sense, single-stranded RNA genome and is a member of theFlaviridae family of viruses. At least six distinct, but relatedgenotypes of HCV, based on phylogenetic analyses, have been identified(Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodesa single polyprotein having more than 3000 amino acid residues (Choo etal., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA(1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991)88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991)88:1711-1715). The polyprotein is processed co- and post-translationallyinto both structural and non-structural (NS) proteins.

In particular, as shown in FIG. 1, several proteins are encoded by theHCV genome. The order and nomenclature of the cleavage products of theHCV polyprotein is as follows:NH₂-C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage of thepolyprotein is catalyzed by host proteases which liberate threestructural proteins, the N-terminal nucleocapsid protein (termed “core”)and two envelope glycoproteins, “E1” (also known as E) and “E2” (alsoknown as E2/NS1), as well as nonstructural (NS) proteins that containthe viral enzymes. The NS regions are termed NS2, NS3, NS4 and NS5. NS2is an integral membrane protein with proteolytic activity and, incombination with NS3, cleaves the NS2-NS3 sissle bond which in turngenerates the NS3 N-terminus and releases a large polyprotein thatincludes both serine protease and RNA helicase activities. The NS3protease serves to process the remaining polyprotein. In thesereactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b andNS5a), and an RNA-dependent RNA polymerase (NS5b). Completion ofpolyprotein maturation is initiated by autocatalytic cleavage at theNS3-NS4a junction, catalyzed by the NS3 serine protease.

E1 is detected as a 32-35 kDa species and is converted into a singleendo H-sensitive band of approximately 18 kDa. By contrast, E2 displaysa complex pattern upon immunoprecipitation consistent with thegeneration of multiple species (Spaete et al., Virol. (1992)188:819-830; Selby et al., J. Virol. (1996) 70:5177-5182; Grakoui etal., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol. (1993)67:4017-4026.). The HCV envelope glycoproteins E1 and E2 form a stablecomplex that is co-immunoprecipitable (Grakoui et al., J. Virol. (1993)67:1385-1395; Lanford et al., Virology (1993) 197:225-235; Ralston etal., J. Virol. (1993) 67:6753-6761).

E1 and E2 are retained within cells and lack complex carbohydrate whenexpressed stably or in a transient Vaccinia virus system (Spaete et al.,Virology (1992) 188:819-830; Ralston et al., J. Virol. (1993)67:6753-6761). Since the E1 and E2 proteins are normally membrane-boundin these expression systems, secreted forms have been produced in orderto facilitate purification of the proteins. See, e.g., U.S. Pat. No.6,121,020. Additionally, intracellular production of E1E2 in Hela cellshas been described. See, e.g., International Publication No. WO98/50556.

The HCV E1 and E2 glycoproteins are of considerable interest becausethey have been shown to be protective against viral challenge in primatestudies. (Choo et al., Proc. Natl. Acad. Sci. USA (1994) 91:1294-1298).However, there remains a need for effective vaccine compositionscomprising these antigens for the prevention of HCV infection.

Vaccine compositions often include immunological adjuvants to enhanceimmune responses. For example, Complete Freund's adjuvant (CFA) is apowerful immunostimulatory agent that has been successfully used withmany antigens on an experimental basis. CFA includes three components: amineral oil, an emulsifying agent, and killed mycobacteria, such asMycobacterium tuberculosis. Aqueous antigen solutions are mixed withthese components to create a water-in-oil emulsion. Although effectiveas an adjuvant, CFA causes severe side-effects, including pain, abscessformation and fever, primarily due to the presence of the mycobacterialcomponent. CFA, therefore, is not used in human and veterinary vaccines.

Muramyl dipeptide (MDP) is the minimal unit of the mycobacterial cellwall complex that generates the adjuvant activity observed with CFA.See, e.g., Ellouz et al., Biochem. Biophys. Res. Commun. (1974) 59:1317.Several synthetic analogs of MDP have been generated that exhibit a widerange of adjuvant potency and side-effects. For a review of theseanalogs, see, Chedid et al., Prog. Allergy (1978) 25:63. Representativeanalogs of MDP include threonyl derivatives of MDP (Byars et al.,Vaccine (1987) 5:223), n-butyl derivatives of MDP (Chedid et al.,Infect. Immun. 35:417), and a lipophilic derivative of a muramyltripeptide (Gisler et al., in Immunomodulations of Microbial Productsand Related Synthetic Compounds (1981) Y. Yamamura and S. Kotani, eds.,Excerpta Medica, Amsterdam, p. 167).

One lipophilic derivative of MDP isN-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE). This muramyl tripeptide includes phospholipid tails that allowassociation of the hydrophobic portion of the molecule with a lipidenvironment while the muramyl peptide portion associates with theaqueous environment. Thus, the MTP-PE itself is able to act as anemulsifying agent to generate stable oil-in-water emulsions. MTP-PE hasbeen used in an emulsion of 4% squalene with 0.008% Tween™ 80, termedMTP-PE-LO (low oil), to deliver the herpes simplex virus gD antigen witheffective results (Sanchez-Pescador et al., J. Immunol. (1988)141:1720-1727), albeit poor physical stability. Recently, MF59, a safe,highly immunogenic, submicron oil-in-water emulsion which contains 4-5%w/v squalene, 0.5% w/v Tween 80™, 0.5% Span 85™, and optionally, varyingamounts of MTP-PE, has been developed for use in vaccine compositions.See, e.g., Ott et al., “MF59—Design and Evaluation of a Safe and PotentAdjuvant for Human Vaccines” in Vaccine Design: The Subunit and AdjuvantApproach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York,1995, pp. 277-296. Choo et al., Proc. Natl. Acad. Sci. USA (1994)91:1294-1298 and Houghton et al., in Viral Hepatitis and Liver Disease(1997), p. 656, describe the use of HCV E1/E2 complexes with submicronoil-in-water emulsions which include MTP-PE.

Bacterial DNA includes unmethylated CpG dinucleotides that haveimmunostimulatory effects on peripheral blood mononuclear cells invitro. Krieg et al., J. Clin. Immunol. (1995) 15:284-292. CpGoligonucleotides have been used to enhance immune responses. See, e.g.,U.S. Pat. Nos. 6,207,646; 6,214,806; 6,218,371; and 6,406,705.

Despite the use of such adjuvants, conventional vaccines often fail toprovide adequate protection against the targeted pathogen. Accordingly,there is a continuing need for effective vaccine compositions againstHCV which include safe and non-toxic adjuvants.

SUMMARY OF THE INVENTION

The present invention is based in part, on the surprising discovery thatthe use of HCV E1E2 antigens, in combination with submicron oil-in-wateremulsions and oligonucleotides containing immunostimulatory nucleic acidsequences (ISS), such as CpY, CpR and unmethylated CpG motifs (acytosine followed by guanosine and linked by a phosphate bond), providesfor significantly higher antibody titers than those observed withoutsuch adjuvants. Alternatively, the compositions herein may be used withISSs alone, without submicron oil-in-water emulsions, or with submicronoil-in-water emulsions alone that lack MTP-PE, without ISSs. The use ofsuch combinations provides a safe and effective approach for enhancingthe immunogenicity of HCV E1E2 antigens.

Accordingly, in one embodiment, the invention is directed to acomposition comprising an HCV E1E2 antigen and a submicron oil-in-wateremulsion that lacks MTP-PE, wherein the submicron oil-in-water emulsionis capable of increasing the immune response to the HCV E1E2 antigen.The composition may further comprise an ISS, such as an oligonucleotidecontaining unmethylated CpG motifs (a “CpG oligonucleotide”), which,when present, acts to enhance the immune response to the antigen.

In yet another embodiment, the subject invention is directed to a methodof stimulating an immune response in a vertebrate subject whichcomprises administering to the subject a therapeutically effectiveamount of an HCV E1E2 antigen and a submicron oil-in-water emulsion thatlacks MTP-PE, wherein the submicron oil-in-water emulsion is capable ofincreasing the immune response to the HCV E1E2 antigen. The subject mayalso be administered one or more ISSs, such as one or moreoligonucleotides containing unmethylated CpG motifs, wherein the ISS iscapable of increasing the immune response to the HCV E1E2 antigen. Thesubmicron oil-in-water emulsion may be present in the same compositionas the antigen or may be administered in a separate composition.Moreover, if an ISS is present, it may be present in the samecomposition as the antigen and/or the submicron oil-in-water emulsion,or in a different composition.

In still further embodiments, the invention is directed to a method ofmaking a composition comprising combining a submicron oil-in-wateremulsion that lacks MTP-PE with an HCV E1E2 antigen. In certainembodiments, the method further comprises combining an ISS, such as anoligonucleotide containing unmethylated CpG motifs capable of increasingthe immune response to the HCV E1E2 antigen, with the E1E2 antigen andthe submicron oil-in-water emulsion.

In additional embodiments, the invention is directed to a compositioncomprising an HCV E1E2 antigen and an ISS, such as a CpG oligonucleotidecapable of increasing the immune response to the HCV E1E2 antigen.

In yet another embodiment, the subject invention is directed to a methodof stimulating an immune response in a vertebrate subject whichcomprises administering to the subject a therapeutically effectiveamount of an HCV E1E2 antigen and an ISS, such as a CpG oligonucleotide,wherein the ISS is capable of increasing the immune response to the HCVE1E2 antigen. The ISS may be present in the same composition as theantigen or may be administered in a separate composition.

In still further embodiments, the invention is directed to a method ofmaking a composition comprising combining an ISS, such as a CpGoligonucleotide, with an HCV E1E2 antigen, wherein the ISS is capable ofincreasing the immune response to the HCV E1E2 antigen.

The CpG molecule in any of the embodiments above may have the formula5′-X₁X₂CGX₃X₄, where X₁ and X₂ are a sequence selected from the groupconsisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT andTpG, and X₃ and X₄ are selected from the group consisting of TpT, CpT,ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, and TpG, wherein “p”signifies a phosphate bond. In certain embodiments, the CpGoligonucleotide comprises the sequence GACGTT, GACGTC, GTCGTT or GTCGCT,flanked by several additional nucleotides.

In an additional embodiment, the CpG oligonucleotide for use in thepresent compositions has the sequence 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ IDNO:1) or the sequence 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:5).

In certain embodiments, the submicron oil-in-water emulsion comprises:

-   -   (1) a metabolizable oil, wherein the oil is present in an amount        of 0.5% to 20% of the total volume and    -   (2) an emulsifying agent, wherein the emulsifying agent is 0.01%        to 2.5% by weight (w/v), and wherein the oil and the emulsifying        agent are present in the form of an oil-in-water emulsion having        oil droplets substantially all of which are about 100 nm to less        than 1 micron in diameter,    -   wherein the submicron oil-in-water emulsion is capable of        increasing the immune response to the HCV E1E2 antigen.

In other embodiments, the submicron oil-in-water emulsion is asdescribed above and lacks any polyoxypropylene-polyoxyethylene blockcopolymer, as well as any muramyl peptide.

In additional embodiments, the emulsifying agent comprises apolyoxyethylene sorbitan mono-, di-, or triester and/or a sorbitanmono-, di-, or triester.

In certain embodiments, the oil is present in an amount of 1% to 12%,such as 1% to 4%, of the total volume and the emulsifying agent is 0.01%to 1% by weight (w/v), such as 0.01% to 0.05% by weight (w/v).

In other embodiments described herein, the submicron oil-in-wateremulsion comprises 4-5% w/v squalene, 0.25-1.0% w/v Tween 80™(polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85™(sorbitan trioleate), and optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE).

In other embodiments, the submicron oil-in-water emulsion consistsessentially of:

-   -   (1) 5% by volume of squalene; and    -   (2) one or more emulsifying agents selected from the group        consisting of Tween 80™ (polyoxyelthylenesorbitan monooleate)        and Span 85™ (sorbitan trioleate), wherein the total amount of        emulsifying agent(s) present is 1% by weight (w/v); wherein the        squalene and the emulsifying agent(s) are present in the form of        an oil-in-water emulsion having oil droplets substantially all        of which are about 100 nm to less than 1 micron in diameter and        wherein the composition lacks any        polyoxypropylene-polyoxyethylene block copolymer, and further        wherein the submicron oil-in-water emulsion is capable of        increasing the immune response to the HCV antigen.

In other embodiments, the one or more emulsifying agents arepolyoxyelthylenesorbitan monooleate and sorbitan trioleate and the totalamount of polyoxyelthylenesorbitan monooleate and sorbitan trioleatepresent is 1% by weight (w/v).

In certain embodiments, the composition lacks a muramyl peptide.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the HCV genome, depicting thevarious regions of the HCV polyprotein.

FIGS. 2A-2C (SEQ ID NOS:3 and 4) shows the nucleotide and correspondingamino acid sequence for the HCV-1 E1/E2/p7 region. The numbers shown inthe figure are relative to the full-length HCV-1 polyprotein. The E1, E2and p7 regions are shown.

FIG. 3 is a diagram of plasmid pMHE1E2-809, encoding E1E2₈₀₉, arepresentative E1 E2 protein for use with the present invention.

FIG. 4 shows E1E2₈₀₉ EIA antibody titers from mice immunized withE1E2₈₀₉ plus CpG; E1E2₈₀₉ plus MF59; E1E2₈₀₉ plus CpG and MF59; andE1E2₈₀₉ plus 4XMF59, as described in the examples. Circles indicateindividual mouse serum antibody titers. Boxes show the geometric meanantibody titer (GMT) of the group of 10 mice. The error bars arecomparison intervals for statistically significant differences asdetermined by one-way analysis of variance.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., FundamentalVirology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.);Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., Blackwell Scientific Publications); T. E. Creighton,Proteins: Structures and Molecular Properties (W.H. Freeman and Company,1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., currentaddition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2ndEdition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an antigen” includes a mixture of two or more antigens,and the like.

The following amino acid abbreviations are used throughout the text:

-   -   Alanine: Ala (A) Arginine: Arg (R)    -   Asparagine: Asn (N) Aspartic acid: Asp (D)    -   Cysteine: Cys (C) Glutamine: Gln (O)    -   Glutamic acid: Glu (E) Glycine: Gly (G)    -   Histidine: His (H) Isoleucine: Ile (I)    -   Leucine: Leu (L) Lysine: Lys (K)    -   Methionine: Met (M) Phenylalanine: Phe (F)    -   Proline: Pro (P) Serine: Ser (S)    -   Threonine: Thr (T) Tryptophan: Trp (W)    -   Tyrosine: Tyr (Y) Valine: Val (V)

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

By an “E1 polypeptide” is meant a molecule derived from an HCV E1region. The mature E1 region of HCV-1 begins at approximately amino acid192 of the polyprotein and continues to approximately amino acid 383,numbered relative to the full-length HCV-1 polyprotein. (See, FIGS. 1and 2A-2C. Amino acids 192-383 of FIGS. 2A-2C correspond to amino acidpositions 20-211 of SEQ ID NO:4.) Amino acids at around 173 throughapproximately 191 (amino acids 1-19 of SEQ ID NO: 4) serve as a signalsequence for E1. Thus, by an “E1 polypeptide” is meant either aprecursor E1 protein, including the signal sequence, or a mature E1polypeptide which lacks this sequence, or even an E1 polypeptide with aheterologous signal sequence. The E1 polypeptide includes a C-terminalmembrane anchor sequence which occurs at approximately amino acidpositions 360-383 (see, International Publication No. WO 96/04301,published Feb. 15, 1996). An E1 polypeptide, as defined herein, may ormay not include the C-terminal anchor sequence or portions thereof.

By an “E2 polypeptide” is meant a molecule derived from an HCV E2region. The mature E2 region of HCV-1 begins at approximately amino acid383-385, numbered relative to the full-length HCV-1 polyprotein. (See,FIGS. 1 and 2A-2C. Amino acids 383-385 of FIGS. 2A-2C correspond toamino acid positions 211-213 of SEQ ID NO:4.) A signal peptide begins atapproximately amino acid 364 of the polyprotein. Thus, by an “E2polypeptide” is meant either a precursor E2 protein, including thesignal sequence, or a mature E2 polypeptide which lacks this sequence,or even an E2 polypeptide with a heterologous signal sequence. The E2polypeptide includes a C-terminal membrane anchor sequence which occursat approximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073). An E2 polypeptide, as defined herein, may or may notinclude the C-terminal anchor sequence or portions thereof. Moreover, anE2 polypeptide may also include all or a portion of the p7 region whichoccurs immediately adjacent to the C-terminus of E2. As shown in FIGS. 1and 2A-2C, the p7 region is found at positions 747-809, numberedrelative to the full-length HCV-1 polyprotein (amino acid positions575-637 of SEQ ID NO:4). Additionally, it is known that multiple speciesof HCV E2 exist (Spaete et al., Virol. (1992) 188:819-830; Selby et al.,J. Virol. (1996) 70:5177-5182; Grakoui et al., J. Virol. (1993)67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026). Accordingly,for purposes of the present invention, the term “E2” encompasses any ofthese species of E2 including, without limitation, species that havedeletions of 1-20 or more of the amino acids from the N-terminus of theE2, such as, e.g, deletions of 1, 2, 3, 4, 5 . . . 10 . . . 15, 16, 17,18, 19 . . . etc. amino acids. Such E2 species include those beginningat amino acid 387, amino acid 402, amino acid 403, etc.

Representative E1 and E2 regions from HCV-1 are shown in FIGS. 2A-2C andSEQ ID NO:4. For purposes of the present invention, the E1 and E2regions are defined with respect to the amino acid number of thepolyprotein encoded by the genome of HCV-1, with the initiatormethionine being designated position 1. See, e.g., Choo et al., Proc.Natl. Acad. Sci. USA (1991) 88:2451-2455. However, it should be notedthat the term an “E1 polypeptide” or an “E2 polypeptide” as used hereinis not limited to the HCV-1 sequence. In this regard, the correspondingE1 or E2 regions in other HCV isolates can be readily determined byaligning sequences from the isolates in a manner that brings thesequences into maximum alignment. This can be performed with any of anumber of computer software packages, such as ALIGN 1.0, available fromthe University of Virginia, Department of Biochemistry (Attn: Dr.William R. Pearson). See, Pearson et al., Proc. Natl. Acad. Sci. USA(1988) 85:2444-2448.

Furthermore, an “E1 polypeptide” or an “E2 polypeptide” as definedherein is not limited to a polypeptide having the exact sequencedepicted in the Figures. Indeed, the HCV genome is in a state ofconstant flux in vivo and contains several variable domains whichexhibit relatively high degrees of variability between isolates. Anumber of conserved and variable regions are known between these strainsand, in general, the amino acid sequences of epitopes derived from theseregions will have a high degree of sequence homology, e.g., amino acidsequence homology of more than 30%, preferably more than 40%, more than60%, and even more than 80-90% homology, when the two sequences arealigned. It is readily apparent that the terms encompass E1 and E2polypeptides from any of the various HCV strains and isolates includingisolates having any of the 6 genotypes of HCV described in Simmonds etal., J. Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.),as well as newly identified isolates, and subtypes of these isolates,such as HCV1a, HCV1b etc.

Thus, for example, the term “E1” or “E2” polypeptide refers to native E1or E2 sequences from any of the various HCV strains, as well as analogs,muteins and immunogenic fragments, as defined further below. Thecomplete genotypes of many of these strains are known. See, e.g., U.S.Pat. No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799.

Additionally, the terms “E1 polypeptide” and “E2 polypeptide” encompassproteins which include modifications to the native sequence, such asinternal deletions, additions and substitutions (generally conservativein nature). These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. All of these modifications areencompassed in the present invention so long as the modified E1 and E2polypeptides function for their intended purpose. Thus, for example, ifthe E1 and/or E2 polypeptides are to be used in vaccine compositions,the modifications must be such that immunological activity (i.e., theability to elicit a humoral or cellular immune response to thepolypeptide) is not lost.

By “E1E2” complex is meant a protein containing at least one E1polypeptide and at least one E2 polypeptide, as described above. Such acomplex may also include all or a portion of the p7 region which occursimmediately adjacent to the C-terminus of E2. As shown in FIGS. 1 and2A-2C, the p7 region is found at positions 747-809, numbered relative tothe full-length HCV-1 polyprotein (amino acid positions 575-637 of SEQID NO:4). A representative E1E2 complex which includes the p7 protein istermed “E1E2₈₀₉” herein.

The mode of association of E1 and E2 in an E1E2 complex is immaterial.The E1 and E2 polypeptides may be associated through non-covalentinteractions such as through electrostatic forces, or by covalent bonds.For example, the E1E2 polypeptides of the present application may be inthe form of a fusion protein which includes an immunogenic E1polypeptide and an immunogenic E2 polypeptide, as defined above. Thefusion may be expressed from a polynucleotide encoding an E1E2 chimera.Alternatively, E1E2 complexes may form spontaneously simply by mixing E1and E2 proteins which have been produced individually. Similarly, whenco-expressed and secreted into media, the E1 and E2 proteins can form acomplex spontaneously. Thus, the term encompasses E1E2 complexes (alsocalled aggregates) that spontaneously form upon purification of E1and/or E2. Such aggregates may include one or more E1 monomers inassociation with one or more E2 monomers. The number of E1 and E2monomers present need not be equal so long as at least one E1 monomerand one E2 monomer are present. Detection of the presence of an E1E2complex is readily determined using standard protein detectiontechniques such as polyacrylamide gel electrophoresis and immunologicaltechniques such as immunoprecipitation.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, or fragments of such derivatives, that retaindesired activity, such as immunoreactivity in assays described herein.In general, the term “analog” refers to compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions (generally conservative in nature) and/ordeletions, relative to the native molecule, so long as the modificationsdo not destroy immunogenic activity. The term “mutein” refers topeptides having one or more peptide mimics (“peptoids”), such as thosedescribed in International Publication No. WO 91/04282. Preferably, theanalog or mutein has at least the same immunoactivity as the nativemolecule. Methods for making polypeptide analogs and muteins are knownin the art and are described further below.

Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25 or50 conservative or non-conservative amino acid substitutions, or anyinteger between 5-50, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

By “fragment” is intended a polypeptide consisting of only a part of theintact full-length polypeptide sequence and structure. The fragment caninclude a C-terminal deletion an N-terminal deletion, and/or an internaldeletion of the native polypeptide. An “immunogenic fragment” of aparticular HCV protein will generally include at least about 5-10contiguous amino acid residues of the full-length molecule, preferablyat least about 15-25 contiguous amino acid residues of the full-lengthmolecule, and most preferably at least about 20-50 or more contiguousamino acid residues of the full-length molecule, that define an epitope,or any integer between 5 amino acids and the full-length sequence,provided that the fragment in question retains the ability to elicit animmunological response as defined herein. For a description of knownimmunogenic fragments of HCV E1 and E2, see, e.g., Chien et al.,International Publication No. WO 93/00365.

The term “epitope” as used herein refers to a sequence of at least about3 to 5, preferably about 5 to 10 or 15, and not more than about 500amino acids (or any integer therebetween), which define a sequence thatby itself or as part of a larger sequence, elicits an immunologicalresponse in the subject to which it is administered. Often, an epitopewill bind to an antibody generated in response to such sequence. Thereis no critical upper limit to the length of the fragment, which maycomprise nearly the full-length of the protein sequence, or even afusion protein comprising two or more epitopes from the HCV polyprotein.An epitope for use in the subject invention is not limited to apolypeptide having the exact sequence of the portion of the parentprotein from which it is derived. Indeed, viral genomes are in a stateof constant flux and contain several variable domains which exhibitrelatively high degrees of variability between isolates. Thus the term“epitope” encompasses sequences identical to the native sequence, aswell as modifications to the native sequence, such as deletions,additions and substitutions (generally conservative in nature).

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA82:178-182; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Using suchtechniques, a number of epitopes of HCV have been identified. See, e.g.,Chien et al., Viral Hepatitis and Liver Disease (1994) pp. 320-324, andfurther below. Similarly, conformational epitopes are readily identifiedby determining spatial conformation of amino acids such as by, e.g.,x-ray crystallography and 2-dimensional nuclear magnetic resonance. See,e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteinscan also be identified using standard antigenicity and hydropathy plots,such as those calculated using, e.g., the Omiga version 1.0 softwareprogram available from the Oxford Molecular Group. This computer programemploys the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA(1981) 78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

As used herein, the term “conformational epitope” refers to a portion ofa full-length protein, or an analog or mutein thereof, having structuralfeatures native to the amino acid sequence encoding the epitope withinthe full-length natural protein. Native structural features include, butare not limited to, glycosylation and three dimensional structure. Thelength of the epitope defining sequence can be subject to widevariations as these epitopes are believed to be formed by thethree-dimensional shape of the antigen (e.g., folding). Thus, aminoacids defining the epitope can be relatively few in number, but widelydispersed along the length of the molecule (or even on differentmolecules in the case of dimers, etc.), being brought into correctepitope conformation via folding. The portions of the antigen betweenthe residues defining the epitope may not be critical to theconformational structure of the epitope. For example, deletion orsubstitution of these intervening sequences may not affect theconformational epitope provided sequences critical to epitopeconformation are maintained (e.g., cysteines involved in disulfidebonding, glycosylation sites, etc.).

Conformational epitopes are readily identified using methods discussedabove. Moreover, the presence or absence of a conformational epitope ina given polypeptide can be readily determined through screening theantigen of interest with an antibody (polyclonal serum or monoclonal tothe conformational epitope) and comparing its reactivity to that of adenatured version of the antigen which retains only linear epitopes (ifany). In such screening using polyclonal antibodies, it may beadvantageous to absorb the polyclonal serum first with the denaturedantigen and see if it retains antibodies to the antigen of interest.Conformational epitopes derived from the E1 and E2 regions are describedin, e.g., International Publication No. WO 94/01778.

An “immunological response” to an HCV antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto molecules present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTLs”). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4+ and CD8+ T-cells. Acomposition or vaccine that elicits a cellular immune response may serveto sensitize a vertebrate subject by the presentation of antigen inassociation with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host. Theability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response, including, or example, neutralizationof binding (NOB) antibodies. The presence of an NOB antibody response isreadily determined by the techniques described in, e.g., Rosa et al.,Proc. Natl. Acad. Sci. USA (1996) 93:1759. Hence, an immunologicalresponse may include one or more of the following effects: theproduction of antibodies by B-cells; and/or the activation of suppressorT-cells and/or 8 T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection oralleviation of symptoms to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

As used herein an “immunostimulatory nucleotide sequence” or “ISS” meansa polynucleotide that includes at least one immunostimulatoryoligonucleotide (ISS-ODN) moiety. The ISS moiety is a single- ordouble-stranded DNA or RNA oligonucleotide having at least sixnucleotide bases that may include, or consist of, a modifiedoligonucleotide or a sequence of modified nucleosides. The ISS moietiescomprise, or may be flanked by, a CG-containing nucleotide sequence or ap(1C) nucleotide sequence, which may be palindromic. The cysteine may bemethylated or unmethylated. Examples of particular ISS molecules for usein the present invention include CpG molecules, discussed further below,as well as CpY and CpR molecules and the like.

A component of an HCV E1E2 composition, such as a submicron oil-in-wateremulsion or CpG oligonucleotide, enhances the immune response to the HCVE1E2 antigen present in the composition when the composition possesses agreater capacity to elicit an immune response than the immune responseelicited by an equivalent amount of the antigen when delivered withoutthe additional component. Such enhanced immunogenicity can be determinedby administering the antigen composition with and without the additionalcomponents, and comparing antibody titers against the two using standardassays such as radioimmunoassay and ELISAs, well known in the art.

A “recombinant” protein is a protein which retains the desired activityand which has been prepared by recombinant DNA techniques as describedherein. In general, the gene of interest is cloned and then expressed intransformed organisms, as described further below. The host organismexpresses the foreign gene to produce the protein under expressionconditions.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

By “equivalent antigenic determinant” is meant an antigenic determinantfrom different sub-species or strains of HCV, such as from strains 1, 2,3, etc., of HCV which antigenic determinants are not necessarilyidentical due to sequence variation, but which occur in equivalentpositions in the HCV sequence in question. In general the amino acidsequences of equivalent antigenic determinants will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than30%, usually more than 40%, such as more than 60%, and even more than80-90% homology, when the two sequences are aligned.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by ═HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

II. Modes of Carrying out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

As noted above, the present invention is based on the discovery that HCVE1E2 antigens, in combination with submicron oil-in-water emulsionslacking MTP-PE, as well as with submicron oil-in-water emulsions andimmunostimulatory nucleic acid molecules, such as CpG oligonucleotides,provide compositions that elicit significantly higher antibody titersthan those observed without such adjuvants. Elicitation of HCV-specificantibodies by E1E2 polypeptides provides both in vitro and in vivo modelsystems for the development of HCV vaccines, particularly foridentifying HCV E1, E2 and HCV E1E2 polypeptide epitopes associated withthe production of strong anti-E1, anti-E2 and/or anti E1E2 antibodytiters, and/or cellular immune responses directed against HCV. E1E2polypeptides can also be used to generate an immune response against anHCV in a mammal, particularly an anti-E1, anti-E2 and/or anti-E1E2antibody response and/or a cellular immune response, for eithertherapeutic or prophylactic purposes.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding E1E2 polypeptides for use in thesubject compositions, as well as production of submicron oil-in-wateremulsions, immunostimulatory nucleic acid molecules and compositionscomprising the above.

E1E2 Polypeptides

As explained above, the E1E2 complexes for use with the presentcompositions comprise E1 and E2 polypeptides, associated either throughnon-covalent or covalent interactions. The genome of the hepatitis Cvirus typically contains a single open reading frame of approximately9,600 nucleotides, which is transcribed into a polyprotein. An HCVpolyprotein is cleaved to produce a number of distinct products, in theorder of NH₂-C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH (see, FIG. 1).The HCV E1 polypeptide is a glycoprotein and extends from approximatelyamino acid 192 to amino acid 383 (numbered relative to the polyproteinof HCV-1). See, Choo et al., Proc. Natl. Acad. Sci. USA (1991)88:2451-2455. Amino acids at around 173 through approximately 191represent a signal sequence for E1. An HCV E2 polypeptide is also aglycoprotein and extends from approximately amino acid 383 or 384 toamino acid 746. A signal peptide for E2 begins at approximately aminoacid 364 of the polyprotein. Thus, the term “full-length” E1 or “nottruncated” E1 as used herein refers to polypeptides that include, atleast, amino acids 192-383 of an HCV polyprotein (numbered relative toHCV-1). With respect to E2, the term “full-length” or “not truncated” asused herein refers to polypeptides that include, at least, amino acids383 or 384 to amino acid 746 of an HCV polyprotein (numbered relative toHCV-1). As will be evident from this disclosure, E2 polypeptides for usewith the present invention may include additional amino acids from thep7 region, such as amino acids 747-809.

As explained above, E2 exists as multiple species (Spaete et al., Virol.(1992) 188:819-830; Selby et al., J. Virol. (1996) 70:5177-5182; Grakouiet al., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol. (1993)67:4017-4026) and clipping and proteolysis may occur at the N- andC-termini of the E1 and E2 polypeptides. Thus, an E2 polypeptide for useherein may comprise at least amino acids 405-661, e.g., 400, 401, 402 .. . to 661, such as 383 or 384-661, 383 or 384-715, 383 or 384-746, 383or 384-749 or 383 or 384-809, or 383 or 384 to any C-terminus between661-809, of an HCV polyprotein, numbered relative to the full-lengthHCV-1 polyprotein. Similarly, preferable E1 polypeptides for use hereincan comprise amino acids 192-326, 192-330, 192-333, 192-360, 192-363,192-383, or 192 to any C-terminus between 326-383, of an HCVpolyprotein.

The E1 E2 complexes may also be made up of immunogenic fragments of E1and E2 which comprise epitopes. For example, fragments of E1polypeptides can comprise from about 5 to nearly the full-length of themolecule, such as 6, 10, 25, 50, 75, 100, 125, 150, 175, 185 or moreamino acids of an E1 polypeptide, or any integer between the statednumbers. Similarly, fragments of E2 polypeptides can comprise 6, 10, 25,50, 75, 100, 150, 200, 250, 300, or 350 amino acids of an E2polypeptide, or any integer between the stated numbers. The E1 and E2polypeptides may be from the same or different HCV strains.

For example, epitopes derived from, e.g., the hypervariable region ofE2, such as a region spanning amino acids 384-410 or 390-410, can beincluded in the E2 polypeptide. A particularly effective E2 epitope toincorporate into the E2 sequence is one which includes a consensussequence derived from this region, such as the consensus sequenceGly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn,which represents a consensus sequence for amino acids 390-410 of the HCVtype 1 genome. Additional epitopes of E1 and E2 are known and describedin, e.g., Chien et al., International Publication No. WO 93/00365.

Moreover, the E1 and E2 polypeptides of the complex may lack all or aportion of the membrane spanning domain. The membrane anchor sequencefunctions to associate the polypeptide to the endoplasmic reticulum.Normally, such polypeptides are capable of secretion into growth mediumin which an organism expressing the protein is cultured. However, asdescribed in International Publication No. WO 98/50556, suchpolypeptides may also be recovered intracellularly. Secretion intogrowth medium is readily determined using a number of detectiontechniques, including, e.g., polyacrylamide gel electrophoresis and thelike, and immunological techniques such as immunoprecipitation assays asdescribed in, e.g., International Publication No. WO 96/04301, publishedFeb. 15, 1996. With E1, generally polypeptides terminating with aboutamino acid position 370 and higher (based on the numbering of HCV-1 E1)will be retained by the ER and hence not secreted into growth media.With E2, polypeptides terminating with about amino acid position 731 andhigher (also based on the numbering of the HCV-1 E2 sequence) will beretained by the ER and not secreted. (See, e.g., InternationalPublication No. WO 96/04301, published Feb. 15, 1996). It should benoted that these amino acid positions are not absolute and may vary tosome degree. Thus, the present invention contemplates the use of E1 andE2 polypeptides which retain the transmembrane binding domain, as wellas polypeptides which lack all or a portion of the transmembrane bindingdomain, including E1 polypeptides terminating at about amino acids 369and lower, and E2 polypeptides, terminating at about amino acids 730 andlower, are intended to be captured by the present invention.Furthermore, the C-terminal truncation can extend beyond thetransmembrane spanning domain towards the N-terminus. Thus, for example,E1 truncations occurring at positions lower than, e.g., 360 and E2truncations occurring at positions lower than, e.g., 715, are alsoencompassed by the present invention. All that is necessary is that thetruncated E1 and E2 polypeptides remain functional for their intendedpurpose. However, particularly preferred truncated E1 constructs arethose that do not extend beyond about amino acid 300. Most preferred arethose terminating at position 360. Preferred truncated E2 constructs arethose with C-terminal truncations that do not extend beyond about aminoacid position 715. Particularly preferred E2 truncations are thosemolecules truncated after any of amino acids 715-730, such as 725. Iftruncated molecules are used, it is preferable to use E1 and E2molecules that are both truncated.

The E1 and E2 polypeptides and complexes thereof may also be present asasialoglycoproteins. Such asialoglycoproteins are produced by methodsknown in the art, such as by using cells in which terminal glycosylationis blocked. When these proteins are expressed in such cells and isolatedby GNA lectin affinity chromatography, the E1 and E2 proteins aggregatespontaneously. Detailed methods for producing these E1E2 aggregates aredescribed in, e.g., U.S. Pat. No. 6,074,852, incorporated herein byreference in its entirety.

Moreover, the E1E2 complexes may be present as a heterogeneous mixtureof molecules, due to clipping and proteolytic cleavage, as describedabove. Thus, a composition including E1E2 complexes may include multiplespecies of E1E2, such as E1E2 terminating at amino acid 746 (E1E2₇₄₆),E1E28 terminating at amino acid 809 (E1E2₈₀₉), or any of the othervarious E1 and E2 molecules described above, such as E2 molecules withN-terminal truncations of from 1-20 amino acids, such as E2 speciesbeginning at amino acid 387, amino acid 402, amino acid 403, etc.

E1 E2 complexes are readily produced recombinantly, either as fusionproteins or by e.g., co-transfecting host cells with constructs encodingfor the E1 and E2 polypeptides of interest. Co-transfection can beaccomplished either in trans or cis, i.e., by using separate vectors orby using a single vector which bears both of the E1 and E2 genes. Ifdone using a single vector, both genes can be driven by a single set ofcontrol elements or, alternatively, the genes can be present on thevector in individual expression cassettes, driven by individual controlelements. Following expression, the E1 and E2 proteins willspontaneously associate. Alternatively, the complexes can be formed bymixing the individual proteins together which have been producedseparately, either in purified or semi-purified form, or even by mixingculture media in which host cells expressing the proteins, have beencultured, if the proteins are secreted. Finally, the E1E2 complexes ofthe present invention may be expressed as a fusion protein wherein thedesired portion of E1 is fused to the desired portion of E2.

Methods for producing E1E2 complexes from full-length, truncated E1 andE2 proteins which are secreted into media, as well as intracellularlyproduced truncated proteins, are known in the art. For example, suchcomplexes may be produced recombinantly, as described in U.S. Pat. No.6,121,020; Ralston et al., J. Virol. (1993) 67:6753-6761, Grakoui etal., J. Virol. (1993) 67:1385-1395; and Lanford et al., Virology (1993)197:225-235.

Thus, polynucleotides encoding HCV E1 and E2 polypeptides for use withthe present invention can be made using standard techniques of molecularbiology. For example, polynucleotide sequences coding for theabove-described molecules can be obtained using recombinant methods,such as by screening cDNA and genomic libraries from cells expressingthe gene, or by deriving the gene from a vector known to include thesame. Furthermore, the desired gene can be isolated directly from viralnucleic acid molecules, using techniques described in the art, such asin Houghton et al., U.S. Pat. No. 5,350,671. The gene of interest canalso be produced synthetically, rather than cloned. The molecules can bedesigned with appropriate codons for the particular sequence. Thecomplete sequence is then assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984)Science 223:1299; and Jay et al. (1984) J. Biol. Chem. 259:6311.

Thus, particular nucleotide sequences can be obtained from vectorsharboring the desired sequences or synthesized completely or in partusing various oligonucleotide synthesis techniques known in the art,such as site-directed mutagenesis and polymerase chain reaction (PCR)techniques where appropriate. See, e.g., Sambrook, supra. In particular,one method of obtaining nucleotide sequences encoding the desiredsequences is by annealing complementary sets of overlapping syntheticoligonucleotides produced in a conventional, automated polynucleotidesynthesizer, followed by ligation with an appropriate DNA ligase andamplification of the ligated nucleotide sequence via PCR. See, e.g.,Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088.Additionally, oligonucleotide directed synthesis (Jones et al. (1986)Nature 54:75-82), oligonucleotide directed mutagenesis of pre-existingnucleotide regions (Riechmann et al. (1988) Nature 332:323-327 andVerhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling-inof gapped oligonucleotides using T₄ DNA polymerase (Queen et al. (1989)Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used to providemolecules having altered or enhanced antigen-binding capabilities andimmunogenicity.

Once coding sequences have been prepared or isolated, such sequences canbe cloned into any suitable vector or replicon. Numerous cloning vectorsare known to those of skill in the art, and the selection of anappropriate cloning vector is a matter of choice. Suitable vectorsinclude, but are not limited to, plasmids, phages, transposons, cosmids,chromosomes or viruses which are capable of replication when associatedwith the proper control elements.

The coding sequence is then placed under the control of suitable controlelements, depending on the system to be used for expression. Thus, thecoding sequence can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator, sothat the DNA sequence of interest is transcribed into RNA by a suitabletransformant. The coding sequence may or may not contain a signalpeptide or leader sequence which can later be removed by the host inpost-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the sequencesrelative to the growth of the host cell. Regulatory sequences are knownto those of skill in the art, and examples include those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory compound.Other types of regulatory elements may also be present in the vector.For example, enhancer elements may be used herein to increase expressionlevels of the constructs. Examples include the SV40 early gene enhancer(Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derivedfrom the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman etal. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived fromhuman CMV (Boshart et al. (1985) Cell 41:521), such as elements includedin the CMV intron A sequence (U.S. Pat. No. 5,688,688). The expressioncassette may further include an origin of replication for autonomousreplication in a suitable host cell, one or more selectable markers, oneor more restriction sites, a potential for high copy number and a strongpromoter.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe molecule of interest may be desirable to achieve this end. Forexample, in some cases it may be necessary to modify the sequence sothat it can be attached to the control sequences in the appropriateorientation; i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

As explained above, it may also be desirable to produce mutants oranalogs of the polypeptide of interest. Mutants or analogs of HCVpolypeptides for use in the subject compositions may be prepared by thedeletion of a portion of the sequence encoding the polypeptide ofinterest, by insertion of a sequence, and/or by substitution of one ormore nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, and the like,are well known to those skilled in the art. See, e.g., Sambrook et al.,supra; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82:448;Geisselsoder et al. (1987) BioTechniques 5:786; Zoller and Smith (1983)Methods Enzymol. 100:468; Dalbie-McFarland et al. (1982) Proc. Natl.Acad. Sci USA 79:6409.

The molecules can be expressed in a wide variety of systems, includinginsect, mammalian, bacterial, viral and yeast expression systems, allwell known in the art.

For example, insect cell expression systems, such as baculovirussystems, are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (“MaxBac” kit). Similarly, bacterialand mammalian cell expression systems are well known in the art anddescribed in, e.g., Sambrook et al., supra. Yeast expression systems arealso known in the art and described in, e.g., Yeast Genetic Engineering(Barr et al., eds., 1989) Butterworths, London.

A number of appropriate host cells for use with the above systems arealso known. For example, mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human embryonic kidney cells, human hepatocellularcarcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”)cells, as well as others. Similarly, bacterial hosts such as E. coli,Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of interest canbe stably integrated into a host cell genome or maintained on a stableepisomal element in a suitable host cell using various gene deliverytechniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346.

Depending on the expression system and host selected, the molecules areproduced by growing host cells transformed by an expression vectordescribed above under conditions whereby the protein is expressed. Theexpressed protein is then isolated from the host cells and purified. Ifthe expression system secretes the protein into growth media, theproduct can be purified directly from the media. If it is not secreted,it can be isolated from cell lysates. The selection of the appropriategrowth conditions and recovery methods are within the skill of the art.

Compositions

Once produced, the E1E2 antigens may be provided in vaccinecompositions, in e.g., prophylactic (i.e., to prevent infection) ortherapeutic (to treat HCV following infection) vaccines. The vaccinescan comprise mixtures of one or more of the E1E2 complexes, such as E1E2complexes derived from more than one viral isolate, as well asadditional HCV antigens. Moreover, as explained above, the E1E2complexes may be present as a heterogeneous mixture of molecules, due toclipping and proteolytic cleavage. Thus, a composition including E1E2complexes may include multiple species of E1E2, such as E1E2 terminatingat amino acid 746 (E1E2₇₄₆), E1E28 terminating at amino acid 809(E1E2₈₀₉), or any of the other various E1 and E2 molecules describedabove, such as E2 molecules with N-terminal truncations of from 1-20amino acids, such as E2 species beginning at amino acid 387, amino acid402, amino acid 403, etc.

The vaccines may be administered in conjunction with other antigens andimmunoregulatory agents, for example, immunoglobulins, cytokines,lymphokines, and chemokines, including but not limited to cytokines suchas IL-2, modified IL-2 (cys125-ser125), GM-CSF, IL-12, γ-interferon,IP-10, MIP1β, FLP-3, ribavirin and RANTES.

The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such vehicles.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycollic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Such carriers are well known to those ofordinary skill in the art. Furthermore, the HCV polypeptide may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc.

As explained herein, submicron oil-in-water emulsions and/or ISSs, suchas CpG oligonucleotides (described further below), may be present in thesame composition to enhance the immune response. Additional adjuvantsmay also be present, such as but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) Ribi™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may beused or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMs may be devoid of additional detergent (see,e.g., International Publication No. WO 00/07621); (4) Complete FreundsAdjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines,such as interleukins, such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12etc. (see, e.g., International Publication No. WO 99/44636),interferons, such as gamma interferon, macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxifiedmutants of a bacterial ADP-ribosylating toxin such as a cholera toxin(CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT),particularly LT-K63 (where lysine is substituted for the wild-type aminoacid at position 63) LT-R72 (where arginine is substituted for thewild-type amino acid at position 72), CT-S 109 (where serine issubstituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202 and WO92/19265); (7)monophosporyl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB2220221; EPA 0689454), optionally in the substantial absence of alum(see, e.g., International Publication No. WO 00/56358); (8) combinationsof 3dMPL with, for example, QS21 and/or oil-in-water emulations (see,e.g., EPA 0835318; EPA 0735898; EPA 0761231); (9) a polyoxyethyleneether or a polyoxyethylene ester (see, e.g., International PublicationNo. WO 99/52549); (10) a saponin and an immunostimulatoryoligonucleotide, such as a CpG oligonucleotide (see, e.g., InternationalPublication No. WO 00/62800); (11) an immunostimulant and a particle ofa metal salt (see, e.g., International Publication No. WO 00/23105);(12) a saponin and an oil-in-water emulsion (see, e.g., InternationalPublication No. WO 99/11241; (13) a saponin (e.g., QS21)+3dMPL+IL-12(optionally+a sterol) (see, e.g., International Publication No. WO98/57659); and (14) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid vehicles prior to injection may also beprepared.

The vaccines will comprise a therapeutically effective amount of theE1E2 complexes and any other of the above-mentioned components, asneeded. By “therapeutically effective amount” is meant an amount of anE1E2 protein which will induce an immunological response, preferably aprotective immunological response, in the individual to which it isadministered. Such a response will generally result in the developmentin the subject of a secretory, cellular and/or antibody-mediated immuneresponse to the vaccine. Usually, such a response includes but is notlimited to one or more of the following effects; the production ofantibodies from any of the immunological classes, such asimmunoglobulins A, D, E, G or M; the proliferation of B and Tlymphocytes; the provision of activation, growth and differentiationsignals to immunological cells; expansion of helper T cell, suppressor Tcell, and/or cytotoxic T cell and/or γδ T cell populations.

Once formulated, the vaccines are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule. Preferably, the effective amountis sufficient to bring about treatment or prevention of diseasesymptoms. The exact amount necessary will vary depending on the subjectbeing treated; the age and general condition of the individual to betreated; the capacity of the individual's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular E1E2 polypeptide selected andits mode of administration, among other factors. An appropriateeffective amount can be readily determined by one of skill in the art. A“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials using in vitro and in vivomodels known in the art. The amount of E1E2 polypeptides used in theexamples below provides general guidance which can be used to optimizethe elicitation of anti-E1, anti-E2 and/or anti-E1E2 antibodies.

In particular, an E1E2 complex is preferably injected intramuscularly toa large mammal, such as a primate, for example, a baboon, chimpanzee, orhuman, at a dose of approximately 0.1 μg to about 5.0 mg per dose, orany amount between the stated ranges, such as 0.5 μg to about 1.0 μg, 1μg to about 500 μg, 2.5 μg to about 250 μg, 4 μg to about 200 μg, suchas 4, 5, 6, 7, 8, 9, 10 . . . 20 . . . 30 . . . 40 . . . 50 . . . 60 . .. 70 . . . 80 . . . 90 . . . 100, etc., μg per dose. E1E2 polypeptidescan be administered either to a mammal that is not infected with an HCVor can be administered to an HCV-infected mammal.

Administration of E1E2 polypeptides can elicit an anti-E1, anti-E2and/or anti-E1E2 antibody titer in the mammal that lasts for at least 1week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year,or longer. E1E2 polypeptides can also be administered to provide amemory response. If such a response is achieved, antibody titers maydecline over time, however exposure to the HCV virus or immunogenresults in the rapid induction of antibodies, e.g., within only a fewdays. Optionally, antibody titers can be maintained in a mammal byproviding one or more booster injections of the E1E2 polypeptides at 2weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1year, or more after the primary injection.

Preferably, an E1E2 polypeptide elicits an antibody titer of at least10, 100, 150, 175, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000,5,000, 10,000, 20,000, 30,000, 40,000, 50,000 (geometric mean titer), orhigher, or any number between the stated titer, as determined using astandard immunoassay, such as the immunoassay described in the examplesbelow. See, e.g., Chien et al., Lancet (1993) 342:933; and Chien et al.,Proc. Natl. Acad. Sci. USA (1992) 89:10011.

Submicron Oil-in-Water Emulsions

As explained above, a submicron oil-in-water emulsion formulation mayalso be administered to the vertebrate subject, either prior to,concurrent with, or subsequent to, delivery of the E1E2 antigen.Submicron oil-in water emulsions for use herein include nontoxic,metabolizable oils and commercial emulsifiers. Examples of nontoxic,metabolizable oils include, without limitation, vegetable oils, fishoils, animal oils or synthetically prepared oils. Fish oils, such as codliver oil, shark liver oils and whale oils, are preferred, withsqualene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene,found in shark liver oil, particularly preferred. The oil component willbe present in an amount of from about 0.5% to about 20% by volume,preferably in an amount up to about 15%, more preferably in an amount offrom about 1% to about 12% and most preferably from 1% to about 4% oil.

The aqueous portion of the adjuvant can be buffered saline orunadulterated water. Since the compositions are intended for parenteraladministration, it is preferable to make up the final solutions so thatthe tonicity, i.e., osmolality, is essentially the same as normalphysiological fluids, in order to prevent post-administration swellingor rapid absorption of the composition due to differential ionconcentrations between the composition and physiological fluids. Ifsaline is used rather than water, it is preferable to buffer the salinein order to maintain a pH compatible with normal physiologicalconditions. Also, in certain instances, it may be necessary to maintainthe pH at a particular level in order to insure the stability of certaincomposition components. Thus, the pH of the compositions will generallybe pH 6-8 and pH can be maintained using any physiologically acceptablebuffer, such as phosphate, acetate, tris, bicarbonate or carbonatebuffers, or the like. The quantity of the aqueous agent present willgenerally be the amount necessary to bring the composition to thedesired final volume.

Emulsifying agents suitable for use in the oil-in-water formulationsinclude, without limitation, sorbitan-based non-ionic surfactants suchas a sorbitan mono-, di-, or triester, for example those commerciallyavailable under the name of Span™ or Arlacel™, such as Span™ 85(sorbitan trioleate); polyoxyethylene sorbitan mono-, di-, or triesterscommercially known by the name Tween™, such as Tween 80™(polyoxyelthylenesorbitan monooleate); polyoxyethylene fatty acidsavailable under the name Myrj™; polyoxyethylene fatty acid ethersderived from lauryl, acetyl, stearyl and oleyl alcohols, such as thoseknown by the name of Brij™; and the like. These substances are readilyavailable from a number of commercial sources, including Sigma, St.Louis, Mo. and ICI America's Inc., Wilmington, Del. These emulsifyingagents may be used alone or in combination. The emulsifying agent willusually be present in an amount of 0.02% to about 2.5% by weight (w/v),preferably 0.05% to about 1%, and most preferably 0.01% to about 0.5.The amount present will generally be about 20-30% of the weight of theoil used.

The emulsions can also contain other immunostimulating agents, such asmuramyl peptides, including, but not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc. Immunostimulating bacterial cell wall components, such asmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), may also be present. Alternatively, the emulsions may befree of these agents, such as free of MTP-PE. The submicron oil-in-wateremulsions of the present invention may also be devoid of anypolyoxypropylene-polyoxyethylene (POP-POE) block copolymers. For adescription of various suitable submicron oil-in-water emulsionformulations for use with the present invention, as well asimmunostimulating agents, see, e.g., International Publication No. WO90/14837; Remington: The Science and Practice of Pharmacy, MackPublishing Company, Easton, Pa., 19th edition, 1995; Van Nest et al.,“Advanced adjuvant formulations for use with recombinant subunitvaccines,” In Vaccines 92, Modern Approaches to New Vaccines (Brown etal., ed.) Cold Spring Harbor Laboratory Press, pp. 57-62 (1992); Ott etal., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for HumanVaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell,M. F. and Newman, M. J. eds.) Plenum Press, New York (1995) pp. 277-296;and U.S. Pat. No. 6,299,884, incorporated herein by reference in itsentirety.

In order to produce submicron particles, i.e., particles less than 1micron in diameter and in the nanometer size range, a number oftechniques can be used. For example, commercial emulsifiers can be usedthat operate by the principle of high shear forces developed by forcingfluids through small apertures under high pressure. Examples ofcommercial emulsifiers include, without limitation, Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), Gaulin Model 30CD(Gaulin, Inc., Everett, Mass.), and Rainnie Minilab Type 8.30H (MiroAtomizer Food and Dairy, Inc., Hudson, Wis.). The appropriate pressurefor use with an individual emulsifier is readily determined by one ofskill in the art. For example, when the Model 110Y microfluidizer isused, operation at 5000 to 30,000 psi produces oil droplets withdiameters of about 100 to 750 nm.

The size of the oil droplets can be varied by changing the ratio ofdetergent to oil (increasing the ratio decreases droplet size),operating pressure (increasing operating pressure reduces droplet size),temperature (increasing temperature decreases droplet size), and addingan amphipathic immunostimulating agent (adding such agents decreasesdroplet size). Actual droplet size will vary with the particulardetergent, oil and immunostimulating agent (if any) and with theparticular operating conditions selected. Droplet size can be verifiedby use of sizing instruments, such as the commercial Sub-Micron ParticleAnalyzer (Model N4MD) manufactured by the Coulter Corporation, and theparameters can be varied using the guidelines set forth above untilsubstantially all droplets are less than 1 micron in diameter,preferably less than about 0.8 microns in diameter, and most preferablyless than about 0.5 microns in diameter. By substantially all is meantat least about 80% (by number), preferably at least about 90%, morepreferably at least about 95%, and most preferably at least about 98%.The particle size distribution is typically Gaussian, so that theaverage diameter is smaller than the stated limits.

Particularly preferred submicron oil-in-water emulsions for use hereinare squalene/water emulsions optionally containing varying amounts ofMTP-PE, such as a submicron oil-in-water emulsions containing 4-5% w/vsqualene, 0.25-1.0% w/v Tween 80™ (polyoxyelthylenesorbitan monooleate),and/or 0.25-1.0% Span 85™ (sorbitan trioleate), and optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), for example, the submicron oil-in-water emulsion known as“MF59” (International Publication No. WO 90/14837; U.S. Pat. No.6,299,884, incorporated herein by reference in its entirety; and Ott etal., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for HumanVaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell,M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp.277-296). MF59 contains 4-5% w/v Squalene (e.g., 4.3%), 0.25-0.5% w/vTween 80™, and 0.5% w/v Span 85™ and optionally contains various amountsof MTP-PE, formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.). Forexample, MTP-PE may be present in an amount of about 0-500 μg/dose, morepreferably 0-250 μg/dose and most preferably, 0-100 μg/dose. As usedherein, the term “MF59-0” refers to the above submicron oil-in-wateremulsion lacking MTP-PE, while the term MF59-MTP denotes a formulationthat contains MTP-PE. For instance, “MF59-100” contains 100 μg MTP-PEper dose, and so on. MF69, another submicron oil-in-water emulsion foruse herein, contains 4.3% w/v squalene, 0.25% w/v Tween 80™, and 0.75%w/v Span 85™ and optionally MTP-PE. Yet another submicron oil-in-wateremulsion is MF75, also known as SAF, containing 10% squalene, 0.4% Tween80™, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidizedinto a submicron emulsion. MF75-MTP denotes an MF75 formulation thatincludes MTP, such as from 100-400 μg MTP-PE per dose.

Submicron oil-in-water emulsions, methods of making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in International Publication No.WO 90/14837 and commonly owned, allowed, U.S. patent application Ser.No. 08/418,870, incorporated herein by reference in its entirety.

Once the submicron oil-in-water emulsion is formulated it can beadministered to the vertebrate subject, either prior to, concurrentwith, or subsequent to, delivery of the antigen, and the ISS, if used.If administered prior to immunization with the antigen, the adjuvantformulations can be administered as early as 5-10 days prior toimmunization, preferably 3-5 days prior to immunization and mostpreferably 1-3 or 2 days prior to immunization with the antigens ofinterest. If administered separately, the submicron oil-in-waterformulation can be delivered either to the same site of delivery as theantigen compositions or to a different delivery site.

If simultaneous delivery is desired, the submicron oil-in-waterformulation can be included with the antigen compositions. Generally,the antigens and submicron oil-in-water emulsion can be combined bysimple mixing, stirring, or shaking. Other techniques, such as passing amixture of the two components rapidly through a small opening (such as ahypodermic needle) can also be used to provide the vaccine compositions.

If combined, the various components of the composition can be present ina wide range of ratios. For example, the antigen and emulsion componentsare typically used in a volume ratio of 1:50 to 50:1, preferably 1:10 to10:1, more preferably from about 1:5 to 3:1, and most preferably about1:1. However, other ratios may be more appropriate for specificpurposes, such as when a particular antigen has a low immungenicity, inwhich case a higher relative amount of the antigen component isrequired.

Immunostimulatory Nucleic Acid Molecules (ISS)

Bacterial DNA has previously been reported to stimulate mammalian immuneresponses. See, e.g., Krieg et al., Nature (1995) 374:546-549. Thisimmunostimulatory ability has been attributed to the high frequency ofimmunostimulatory nucleic acid molecules (ISSs), such as unmethylatedCpG dinucleotides present in bacterial DNA. Oligonucleotides containingunmethylated CpG motifs have been shown to induce activation of B cells,NK cells and antigen-presenting cells (APCs), such as monocytes andmacrophages. See, e.g., U.S. Pat. No. 6,207,646.

The present invention makes use of adjuvants derived from ISSs. The ISSof the invention includes an oligonucleotide which can be part of alarger nucleotide construct such as plasmid or bacterial DNA. Theoligonucleotide can be linearly or circularly configured, or can containboth linear and circular segments. The oligonucleotide may includemodifications such as, but are not limited to, modifications of the YOHor 5′OH group, modifications of the nucleotide base, modifications ofthe sugar component, and modifications of the phosphate group. The ISScan comprise ribonucleotides (containing ribose as the only or principalsugar component), deoxyribonucleotides (containing deoxyribose as theprincipal sugar component). Modified sugars or sugar analogs may also beincorporated in the oligonucleotide. Examples of sugar moieties that canbe used include ribose, deoxyribose, pentose, deoxypentose, hexose,deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar analogcyclopentyl group. The sugar may be in pyranosyl or in a furanosyl form.A phosphorous derivative (or modified phosphate group) can be used andcan be a monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphoronthioate, phosphorodithioate, or the like.Nucleic acid bases that are incorporated in the oligonucleotide base ofthe ISS can be naturally occurring purine and pyrimidine bases, namely,uracil or thymine, cytosine, adenine and guanine, as well as naturallyoccurring and synthetic modifications of these bases. Moreover, a largenumber of non-natural nucleosides comprising various heterocyclic basesand various sugar moieties (and sugar analogs) are available, and knownto those of skill in the art.

Structurally, the root oligonucleotide of the ISS is a CG-containingnucleotide sequence or a p(1C) nucleotide sequence, which may bepalindromic. The cytosine may be methylated or unmethylated. Examples ofparticular ISS molecules for use in the present invention include CpG,CpY and CpR molecules, and the like, known in the art.

Preferred ISSs are those derived from the CpG family of molecules, CpGdinucleotides and synthetic oligonucleotides which comprise CpG motifs(see, e.g., Krieg et al. Nature (1995) 374:546 and Davis et al. J.Immunol. (1998) 160:870-876), such as any of the variousimmunostimulatory CpG oligonucleotides disclosed in U.S. Pat. No.6,207,646, incorporated herein by reference in its entirety. Such CpGoligonucleotides generally comprise at least 8 up to about 100nucleotides, preferably 8 to 40 nucleotides, more preferably 15-35nucleotides, preferably 15-25 nucleotides, and any number of nucleotidesbetween these values. For example, oligonucleotides comprising theconsensus CpG motif, represented by the formula 5′-X₁CGX₂-3′, where X₁and X₂ are nucleotides and C is unmethylated, will find use asimmunostimulatory CpG molecules. Generally, X₁ is A, G or T, and X₂ is Cor T. Other useful CpG molecules include those captured by the formula5′-X₁X₂CGX₃X₄, where X, and X₂ are a sequence such as GpT, GpG, GpA,ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT or TpG, and X₃ and X₄ are TpT,CpT, ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, or TpG, wherein“p” signifies a phosphate bond. Preferably, the oligonucleotides do notinclude a GCG sequence at or near the 5′- and/or 3′ terminus.Additionally, the CpG is preferably flanked on its 5′-end with twopurines (preferably a GpA dinucleotide) or with a purine and apyrimidine (preferably, GpT), and flanked on its 3′-end with twopyrimidines, preferably a TpT or TpC dinucleotide. Thus, preferredmolecules will comprise the sequence GACGTT, GACGTC, GTCGTT or GTCGCT,and these sequences will be flanked by several additional nucleotides,such as with 1-20 or more nucleotides, preferably 2 to 10 nucleotidesand more preferably, 3 to 5 nucleotides, or any integer between thesestated ranges. The nucleotides outside of the central core area appearto be extremely amendable to change.

Moreover, the CpG oligonucleotides for use herein may be double- orsingle-stranded. Double-stranded molecules are more stable in vivo whilesingle-stranded molecules display enhanced immune activity.Additionally, the phosphate backbone may be modified, such asphosphorodithioate-modified, in order to enhance the immunostimulatoryactivity of the CpG molecule. As described in U.S. Pat. No. 6,207,646,CpG molecules with phosphorothioate backbones preferentially activateB-cells, while those having phosphodiester backbones preferentiallyactivate monocytic (macrophages, dendritic cells and monocytes) and NKcells.

Exemplary CpG oligonucleotides for use in the present compositionsinclude molecules with the sequence 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ IDNO:1) and 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:5).

ISS molecules can readily be tested for their ability to stimulate animmune response using standard techniques, well known in the art. Forexample, the ability of the molecule to stimulate a humoral and/orcellular immune response is readily determined using the immunoassaysdescribed above. Moreover, the antigen and submicron oil-in-watercompositions can be administered with and without the ISSs to determinewhether an immune response is enhanced.

As explained above, the ISS can be administered either prior to,concurrent with, or subsequent to, delivery of the antigen and/or thesubmicron oil-in-water emulsion. If administered prior to immunizationwith the antigen and/or the submicron oil-in-water emulsion, the ISS canbe administered as early as 5-10 days prior to immunization, preferably3-5 days prior to immunization and most preferably 1-3 or 2 days priorto immunization. If administered separately, the ISS can be deliveredeither to the same site of delivery as the antigen compositions or to adifferent delivery site. If simultaneous delivery is desired, the ISScan be included with the antigen compositions.

Generally about 0.5 μg to 5000 μg of the ISS will be used, moregenerally 0.5 μg to about 1000, preferably 0.5 μg to about 500 μg, orfrom 1 to about 100 μg, preferably about 5 to about 50 μg, preferably 5to about 30, or any amount within these ranges, of the ISS per dose,will find use with the present methods.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va., under the provisions of the Budapest Treaty.The accession number indicated was assigned after successful viabilitytesting, and the requisite fees were paid. The designated deposits willbe maintained for a period of thirty (30) years from the date ofdeposit, or for five (5) years after the last request for the deposit,whichever is longer. Should a culture become nonviable or beinadvertently destroyed, or, in the case of plasmid-containing strains,lose its plasmid, it will be replaced with a viable culture(s) of thesame taxonomic description.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35USC §112. Should there be a discrepancy between the sequence presentedin the present application and the sequence of the gene of interest inthe deposited plasmid due to routine sequencing errors, the sequence inthe deposited plasmid controls. A license may be required to make, use,or sell the deposited materials, and no such license is hereby granted.Strain Deposit Date ATCC No. E1E2₈₀₉ in CHO cells Aug. 16, 2001 PTA-3643

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Production of HCV E1E2

An HCV E1E2 complex for use in the present vaccine compositions wasprepared as a fusion protein as follows. In particular, mammalianexpression plasmid pMH-E1E2-809 (FIG. 3; ATCC Accession No. PTA 3643)encodes an E1E2 fusion protein which includes amino acids 192-809 ofHCV-1 (see, Choo et al., Proc. Natl. Acad. Sci. USA (1991)88:2451-2455). The sequence of the E1E2₈₀₉ molecule is shown in FIGS.2A-2C herein.

Chinese Hamster Ovary (CHO) cells were used for expression of the HCVE1E2 sequence from pMH-E1E2-809. In particular, CHO DG44 cells wereused. These cells, described by Uraub et al., Proc. Natl. Acad. Sci. USA(1980) 77:4216-4220, were derived from CHO K-1 cells and were madedihydrofolate reductase (dhfr) deficient by virtue of a double deletionin the dhfr gene.

DG44 cells were transfected with pMH-E1E2-809. The transfected cellswere grown in selective medium such that only those cells expressing thedhfr gene could grow (Sambrook et al., supra). Isolated CHO colonieswere picked (˜800 colonies) into individual wells of a 96-well plate.From the original 96-well plates, replicates were made to performexpression experiments. The replicate plates were grown until the cellsmade a confluent monolayer. The cells were fixed to the wells of theplate and permeablized using cold methanol. 3D5C3, a monoclonal antibodyagainst E1E2, and 3E5-1 a monoclonal antibody against E2, were used toprobe the fixed cells. After adding an anti-mouse HRP conjugate,followed by substrate, the cell lines with the highest expression weredetermined. The highest expressing cell lines were then expanded to24-well cluster plates. The assay for expression was repeated, andagain, the highest expressing cell lines were expanded to wells ofgreater volume. This was repeated until the highest expressing celllines were expanded from 6-well plates into tissue culture flasks. Atthis point there was sufficient quantity of cells to allow accuratecount and harvest of the cells, and quantitative expression assays weredone. An ELISA (Spaete et al., Virol (1992) 188:819-830) was performedon the cell extract, to determine high expressors.

EXAMPLE 2 Purification of HCV E1E2

Following expression, CHO cells were lysed and the intracellularlyproduced E1E2₈₀₉ was purified by GNA-lectin affinity chromatography (GNAstep), followed by hydroxyapatite (HAP) column chromatography (HAPstep), DV50 membrane filtration (DV50 step), SP Sepharose HP columnchromatography (SP step), Q membrane filtration (Q step) and G25Sephadex column chromatography G25 step). At the completion of each ofthe processing steps, the product pool was either 0.2μ filtered and heldat 2-8° C. or processed immediately through the next purification step.At the completion of the purification process, the antigen was 0.2μfiltered and held frozen at −60° C., or lower until filtered forformulation.

Specifically, to lyse the cells, two volumes of chilled lysis buffer (1%Triton X-100 in 100 mM Tris, pH8, and 1 mM EDTA) were added to the CHOcells at 2-8° C. The mixture was centrifuged at 5000 rpm for 45 min at2-8° C. to remove debris. The supernatant was collected and filteredthrough a Sartorias 0.65 μm Sartopure prefilter (Sartorius) then aSartorias 0.65 mm Sartofine prefilter, followed by a Sartorious 0.45 μmSartobran filter and a 0.2 μm Sartobran filter. The filtered lysate waskept on ice prior to loading on the GNA column.

A GNA agarose column (1885 ml, 200×600, Vector Labs, Burlingame, Calif.)was pre-equilibrated with eight column volumes of equilibration buffer(25 mM NaPO₄, 1.0 M NaCl, 12% Triton X-100, pH 6.8) prior to loading.The lysate was applied to the column at 31.4 ml/min (6 cm/hr) overnight. The column was washed with 4 bed volumes of equilibration buffer,then washed again with 5 bed volumes of 10 mM NaPO₄, 80 mM NaCl, 0.1%Triton X-100, pH 6.8. The product was eluted with 1 M methylα-D-mannopyranoside (MMP), 10 mM NaPO₄, 80 mM NaCl, 0.1% Triton X-100,pH 6.8. The elution peak, about 1 column volume, was collected, 02 μmfiltered and stored at or below −60° C. for HAP chromatography.

HAP chromatography was conducted at room temperature. A 1200 ml (100×150mm) type I ceramic hydroxyapatite column (BioRad) was conditioned withone column volume of 0.4 M NaPO₄, pH 6.8, then equilibrated with notless than ten column volumes of 10 mM NaPO₄, 80 mM NaCl, 0.1% TritonX-100, pH 6.8. Four lots of GNA eluate pools were thawed in acirculating water bath at not more than 30° C., 0.2 μm filtered andloaded onto the equilibrated column at 131 ml/min (100 cm/hr). HAPequilibration buffer was applied to the column as a chase bufferfollowing the load. The flow-through was collected when UV rose abovebaseline. The product collection was stopped when the product poolvolume reached to a volume of load volume plus 75% of the column volume.The HAP flow-through pool was further processed by DV50 viral reductionfiltration.

DV50 Filtration was conducted at room temperature. DV50 load wasprepared by diluting the HAP pool two-fold and adjusting to 0.15% TritonX-100, 1 mM EDTA, pH 5.3. Dilution and adjustment were achieved byadding Dilution Buffer-1 (3 mM citric acid, 2 mM EDTA, 0.2% TritonX-100) to adjust the pH of the product pool to 5.3, followed by additionof Dilution Buffer-2 (2 mM EDTA, 0.2% Triton X-100, pH 5.3) to bring thefinal volume to 2-fold of the original HAP pool volume.

The diluted and adjusted HAP pool (DV50 Load) was filtered through a10-inch, Pall Ultipor VF DV50 membrane cartridge (Pall). The filterhousing was assembled with filter cartridge, prewetted with water, andsterilized by autoclaving at 123° C. for 60 minutes with slow exhaustprior to use. The filter was then prewetted with SP equilibration buffer(10 mM Sodium Citrate, 1 mM EDTA, 0.15% Triton X-100, pH 5.3), anddrained before application of the DV50 load at a pressure not more than45 psi. DV50 load was subsequently applied with a flux rate of about 800ml/min at a transmembrane pressure of about 30 psi. The filtrate wascollected and stored at 2-8° C. overnight and used in the SP step.

SP chromatography was conducted at room temperature in room. An 88-ml(50×45 mm) SP Sepharose HP column (Pharmacia, Peapack, N.J.) wasequilibrated with 15 column volumes of equilibration buffer (10 mMSodium Citrate, 1 mM EDTA, 0.15% Triton X-100, pH 5.3). The DV50filtrate was applied to the column. The column was washed first with 5column volumes of equilibration buffer followed by 20 column volumes ofwash buffer containing 10 mM Sodium Citrate, 15 mM NaCl, 1 mM EDTA, 0.1%Tween-80™, pH 6.0. Product was eluted from the column with 10 mM SodiumCitrate, 180 mM NaCl, 1 mM EDTA, 0.1% Tween-80™, pH 6.0. The entire 280nm absorption peak was collected as product pool. The product pool wasstored at 2-8° C. overnight and used in the Q-membrane filtration step.

The Q-membrane filtration step was conducted at room temperature. Twosterilized Sartorious Q100× disc membranes were connected in series. Themembranes were equilibrated with not less than 300 ml of Q equilibrationbuffer (10 mM Sodium Citrate, 180 mM NaCl, 1 mM EDTA, 0.1% Tween-80™, pH6.0). The entire SP eluate pool was filtered through equilibrated Qmembranes at a flow rate of 30-100 ml/min, followed by flushing with 40ml of Q equilibration buffer. The filtrate and the flush were collectedand combined as the product pool and used in the G25 step.

The G25 step was conducted at room temperature. A 1115-ml (100×142 mm)Pharmacia Sephadex G-25 column (Pharmacia, Peapack, N.J.) wasequilibrated with not less than five column volumes of formulationbuffer (10 mM Sodium Citrate, 270 mM NaCl, 1 mM EDTA, 0.1% Tween-80™, pH6.0). Q filtrate pool was applied to the column and the columnflow-through collected, filtered through a 0.22 μm filter (Millipore)and stored frozen at −60° C. or below, until use.

EXAMPLE 3 Immunogenicity of HCV E1E2 Vaccine Compositions in Mice

The immunogenicity of HCV E1E2₈₀₉, produced and purified as describedabove, in combination with a submicron oil-in-water emulsion and/or aCpG oligonucleotide, was determined as follows.

The formulations used in this study are summarized in Table 1. MF59, asubmicron oil-in-water emulsion which contains 4-5% w/v squalene, 0.5%w/v Tween 80™, 0.5% Span 85™, was produced as described previously. See,International Publication No. WO 90/14837; U.S. Pat. No. 6,299,884,incorporated herein by reference in its entirety; and Ott et al.,“MF59—Design and Evaluation of a Safe and Potent Adjuvant for HumanVaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell,M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296.For groups 4 and 9, four times the amount of MF59 was used. The MF59used in this study was MF59-0, and did not contain any MTP-PE.

The formulations used for groups 1, 3, 6 and 8 also included 25 μg of anactive CpG molecule per dose. The sequence of the active CpG moleculeused was: 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO:1).

The formulation used for group 5 included 25 μg of an inactive controlCpG molecule per dose. The sequence of the inactive CpG molecule usedwas: 5′-TCCAGGACTTCTCTCAGGTT-3′ (SEQ ID NO:2).

The formulations used for groups 1-4 included 2.8 μg per dose of the HCVE1E2₈₀₉ antigen, produced as described above.

The formulations used for groups 5-9 included 2.0 μg per dose of HCVE2₇₁₅, a truncated E2 protein, produced in CHO cells, as described inU.S. Pat. No. 6,12,020.

Balb/C mice, six weeks of age, were divided into 9 groups (10 mice pergroup) and administered, intramuscularly 50 μl of a vaccine compositionwith the components specified in Table 1. Animals were boosted at 30 and90 days following the initial injection. Serum was collected 14 daysfollowing the last injection and anti-E1E2 and anti-E2 antibody titersdetermined by enzyme immunoassays. See, Chien et al., Lancet (1993)342:933.

The results are shown in Table 1 and FIG. 4. As can be seen, miceimmunized with HCV E1E2 using CpG combined with MF59 as adjuvant,produced significantly higher (P<0.05) levels of E1E2 antibodies thanmice immunized with E1E2 using MF59 alone or 4xMF59 alone as adjuvants.CpG alone produced antibody levels higher than antibody levels with MF59alone, albeit, not significantly higher. In contrast, mice immunizedwith E2₇₁₅ using MF59 and/or CpG, produced very low levels of antibodieswith less than 50% of the mice responding. This is surprising asprevious experiments with E2₇₁₅ have produced high antibody levels inmice, with all mice tested responding. TABLE 1 Immunogenicity of HCVE1E2₈₀₉ and E2₇₁₅ using CPG and or MF59 as adjuvants. The numbers inparenthesis indicate the number of animals producing antibodies relativeto the number of animals immunized. Geometric Mean E1E2 GeometricVaccine; EIA Antibody Mean E2 EIA Group Adjuvant Dose Titer AntibodyTiter 1 E1E2₈₀₉; 2.8, 2.8, 2.8  5,167 ND CpG (10/10) 2 E1E2₈₀₉; 2.8,2.8, 2.8  2,716 ND MF59 (10/10) 3 E1E2₈₀₉; 2.8, 2.8, 2.8 19,159^(B) NDCpG + MF59 (10/10) P < 0.05 4 E1E2₈₀₉; 2.8, 2.8, 2.8  3,335 ND 4X MF59(10/10) 5 E2₇₁₅; 2.0, 2.0, 2.0 ND 1.3 Control CpG (1/10) 6 E2₇₁₅; 2.0,2.0, 2.0 ND 3.1 CpG (2/20) 7 E2₇₁₅; 2.0, 2.0, 2.0 ND 6.1 MF59 (4/10) 8E2₇₁₅; 2.0, 2.0, 2.0 ND 26.8  CpG + MF59 (5/10) 9 E2₇₁₅; 2.0, 2.0, 2.0ND 9.7 4xMF59 (4/10)

EXAMPLE 4 Immunogenicity of HCV E1E2 Vaccine Compositions in Chimpanzees

The immunogenicity of HCV E1E2₈₀₉, produced and purified as describedabove, in combination with a submicron oil-in-water emulsion and/or aCpG oligonucleotide, was determined as follows.

The formulations used in this study are summarized in Table 2. MF59 andE1E2₈₀₉ are described above. The sequence of the CpG molecule used was:5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:5).

Chimpanzees were divided into 2 groups (5 animals per group) andadministered, intramuscularly a vaccine composition with the componentsspecified in Table 1. In particular, one group of animals was immunizedat 0, 1 and 6 months with 20 μg of E1E2₈₀₉ and MF59. The second group ofanimals was also immunized at 0, 1 and 6 months with 20 fig of E1E2₈₀₉and MF59, as well as with 500 μg CpG.

Serum samples were obtained 14 days after the last immunization andanti-E1E2 antibody titers determined by enzyme immunoassays. Inparticular, the E1E2 antigen was coated on polystyrene microtiter platesand bound antibody was detected with a HRP-conjugated anti-humanantibody followed by tetramethylbenzidine substrate development.

As can be seen in Table 2, chimpanzees immunized with HCV E1E2 using CpGcombined with MF59 as adjuvant, produced significantly higher (P<0.05)levels of E1E2 antibodies than animals immunized with E1E2 using MF59alone. TABLE 2 Immunogenicity of HCV E1E2₈₀₉ using CPG and MF59 asadjuvants. Geometric Mean E1E2 Vaccine; E1E2 EIA EIA Antibody AdjuvantChimp Antibody Titer Titer Group 1: 1 84 261 E1E2₈₀₉; 2 101 CpG 3 131 4421 5 2580 Group 2: 1 8835 2713 E1E2₈₀₉; 2 2713 CpG + MF59 3 3201 4 5105 1238

Accordingly, novel HCV vaccine compositions and methods of using thesame are disclosed. From the foregoing, it will be appreciated that,although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A composition comprising: (a) a hepatitis C virus (HCV) envelopeantigen; (b) a submicron oil-in-water emulsion that lacksN-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE); and (c) an immunostimulatory nucleic acid sequence (ISS). 2.The composition of claim 1, wherein the HCV envelope antigen comprisesHCV E1E2 complexes.
 3. The composition of claim 2, wherein at least oneof the HCV E1E2 complexes consists of: (a) amino acids 20-210 of SEQ IDNO:4 or sequence of amino acids with at least 80% sequence identity tothe contiguous sequence of amino acids 20-210 of SEQ ID NO:4; (b) aminoacids 211-574 of SEQ ID NO:4 or sequence of amino acids with at least80% sequence identity to the contiguous sequence of amino acids 211-574of SEQ ID NO:4; and (c) amino acids 575-637 of SEQ ID NO:4 or sequenceof amino acids with at least 80% sequence identity to the contiguoussequence of amino acids 575-637 of SEQ ID No:4.
 4. The composition ofclaim 3, wherein at least one of the HCV E1E2 antigen complexes consistsof: (a) amino acids 20-210 of SEQ ID NO:4; (b) amino acids 211-574 ofSEQ ID NO:4; and (c) amino acids 575-637 of SEQ ID NO:4.
 5. Thecomposition of claim 1, wherein the ISS is a CpG oligonucleotide.
 6. Thecomposition of claim 5, wherein the CpG oligonucleotide comprises thesequence 5′-X₁X₂CGX₃×4, where X₁ and X₂ are a sequence selected from thegroup consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA,TpT and TpG; and X₃ and X₄ are selected from the group consisting ofTpT, CpT, ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, and TpG,wherein p signifies a phosphate bond.
 7. The composition of claim 5,wherein the CpG oligonucleotide comprises the sequence GACGTT, GACGTC,GTCGTT or GTCGCT.
 8. The composition of claim 7, wherein the CpGoligonucleotide comprises the sequence 5′-TCCATGACGTTCCTGACGTT-3′(SEQ IDNO:1).
 9. The composition of claim 7, wherein the CpG oligonucleotidecomprises the sequence 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′(SEQ ID NO:5). 10.The composition of claim 1, wherein the submicron oil-in-water emulsioncomprises: (1) a metabolizable oil, wherein the oil is present in anamount of 0.5% to 20% of the total volume; and (2) an emulsifying agent,wherein the emulsifying agent is present in an amount of 0.01% to 2.5%by weight (w/v), and wherein the oil and the emulsifying agent arepresent in the form of an oil-in-water emulsion having oil dropletssubstantially all of which are about 100 nm to less than 1 micron indiameter.
 11. The composition of claim 10, wherein the oil is present inan amount of 1% to 12% of the total volume and the emulsifying agent ispresent in an amount of 0.01% to 1% by weight (w/v).
 12. The compositionof claim 10, wherein the emulsifying agent comprises a polyoxyethylenesorbitan mono-, di-, or triester and/or a sorbitan mono-, di-, ortriester.
 13. The composition of claim 10, wherein the submicronoil-in-water emulsion comprises 4-5% w/v squalene, 0.25-1.0% w/vpolyoxyelthylenesorbitan monooleate, and/or 0.25-1.0% sorbitantrioleate.
 14. The composition of claim 13, wherein the submicronoil-in-water emulsion consists essentially of 5% by volume of squalene;and one or more emulsifying agents selected from the group consisting ofpolyoxyelthylenesorbitan monooleate and sorbitan trioleate, wherein thetotal amount of emulsifying agent(s) present is 1% by weight (w/v). 15.The composition of claim 14, wherein the one or more emulsifying agentsare polyoxyelthylenesorbitan monooleate and sorbitan trioleate and thetotal amount of polyoxyelthylenesorbitan monooleate and sorbitantrioleate present is 1% by weight (w/v).
 16. A method of stimulating animmune response in a vertebrate subject which comprises administering tothe subject a therapeutically effective amount of a hepatitis C virus(HCV) envelope antigen, a submicron oil-in-water emulsion that lacksN-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), and an immunostimulatory nucleic acid sequence (ISS).
 17. Themethod of claim 16, wherein the HCV envelope antigen comprises HCV E1E2complexes.
 18. The method of claim 17, wherein at least one of the HCVE1E2 complexes consists of: (a) amino acids 20-210 of SEQ ID NO:4 orsequence of amino acids with at least 80% sequence identity to thecontiguous sequence of amino acids 20-210 of SEQ ID NO:4; (b) aminoacids 211-574 of SEQ ID NO:4 or sequence of amino acids with at least80% sequence identity to the contiguous sequence of amino acids 211-574of SEQ ID NO:4; and (c) amino acids 575-637 of SEQ ID NO:4 or sequenceof amino acids with at least 80% sequence identity to the contiguoussequence of amino acids 575-637 of SEQ ID NO:4.
 19. The method of claim18, wherein at least one of the HCV E1 E2 antigen complexes consists of:(a) amino acids 20-210 of SEQ ID NO:4; (b) amino acids 211-574 of SEQ IDNO:4; and (c) amino acids 575-637 of SEQ ID NO:4.
 20. The method ofclaim 16, wherein the submicron oil-in-water emulsion is present in thesame composition as the antigen.
 21. The method of claim 16, wherein theISS is a CpG oligonucleotide.
 22. The method of claim 21, wherein theCpG oligonucleotide comprises the sequence 5′-X₁X₂CGX₃×4, where X₁ andX₂ are a sequence selected from the group consisting of GpT, GpG, GpA,ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT and TpG; and X₃ and X₄ areselected from the group consisting of TpT, CpT, ApT, ApG, CpG, TpC, ApC,CpC, TpA, ApA, GpT, CpA, and TpG, wherein p signifies a phosphate bond.23. The method of claim 21, wherein the CpG oligonucleotide comprisesthe sequence GACGTT, GACGTC, GTCGTT or GTCGCT.
 24. The method of claim23, wherein the CpG oligonucleotide comprises the sequence5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO:1).
 25. The method of claim 23,wherein the CpG oligonucleotide comprises the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:5).
 26. The method of claim16, wherein the submicron oil-in-water emulsion comprises: (1) ametabolizable oil, wherein the oil is present in an amount of 0.5% to20% of the total volume; and (2) an emulsifying agent, wherein theemulsifying agent is present in an amount of 0.01% to 2.5% by weight(w/v), and wherein the oil and the emulsifying agent are present in theform of an oil-in-water emulsion having oil droplets substantially allof which are about 100 nm to less than 1 micron in diameter.
 27. Themethod of claim 26, wherein the oil is present in an amount of 1% to 12%of the total volume and the emulsifying agent is present in an amount of0.01% to 1% by weight (w/v).
 28. The method of claim 26, wherein theemulsifying agent comprises a polyoxyethylene sorbitan mono-, di-, ortriester and/or a sorbitan mono-, di-, or triester.
 29. The method ofclaim 28, wherein the submicron oil-in-water emulsion comprises 4-5% w/vsqualene, 0.25-1.0% w/v polyoxyelthylenesorbitan monooleate, and/or0.25-1.0% sorbitan trioleate.
 30. The method of claim 29, wherein thesubmicron oil-in-water emulsion consists essentially of 5% by volume ofsqualene; and one or more emulsifying agents selected from the groupconsisting of polyoxyelthylenesorbitan monooleate and sorbitantrioleate, wherein the total amount of emulsifying agent(s) present is1% by weight (w/v).
 31. The method of claim 30, wherein the one or moreemulsifying agents are polyoxyelthylenesorbitan monooleate and sorbitantrioleate and the total amount of polyoxyelthylenesorbitan monooleateand sorbitan trioleate present is 1% by weight (w/v).
 32. A method ofmaking a composition comprising combining a submicron oil-in-wateremulsion that lacksN-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), with a hepatitis C virus (HCV) envelope antigen and animmunostimulatory nucleic acid sequence (ISS).